How have introns evolved? Did they occur early in the prebiotic world and contribute to the formation of ancient genes by "exon shuffling," or have they been inserted into genes as mobile genetic elements throughout evolution? Important insights into intron evolution have come from analysis of the chloroplast genome of the photosynthetic protist Euglena gracilis, which contains at least 120 introns, several introns-within-introns, known as "twintrons", and complex twintrons formed from 3 or 4 individual introns which are sequentially spliced. A detailed study of the group III introns and twintrons of Euglena is proposed. The specific aims include: (1) Characterization of additional group III introns and twintrons and their RNA processing pathways, (2) Determination of the extent and importance of alternative splicing of group III introns, and specifically whether alternative splicing results in alternative polypeptides products from a single gene, (3) A study of how group III introns and twintrons have evolved, addressed through a comparative analysis of group III introns in species related to Euglena gracilis, (4) Studies on a gene within a twintron, orf458 of the psbC locus, to assess if this gene may have a role in the mobility or splicing of group III introns, (5) Development of a system for transformation of Euglena chloroplasts, in order to study cis- and trans-acting factors in intron and twintron splicing in transgenic plastids, and (6) Attempts to develop an in vitro group III intron splicing system. This work is addressed at understanding fundamental question about the origins of introns, splicing mechanisms, and alternative splicing.